A bioprinted complex tissue model for myotendinous junction with biochemical and biophysical cues

Abstract In the musculoskeletal system, the myotendinous junction (MTJ) is optimally designed from the aspect of force transmission generated from a muscle through a tendon onto the bone to induce movement. Although the MTJ is a key complex tissue in force transmission, the realistic fabrication, and formation of complex tissues can be limited. To obtain the MTJ construct, we prepared two bioinks, muscle‐ and tendon‐derived decellularized extracellular matrix (dECM), which can induce myogenic and tenogenic differentiation of human adipose‐derived stem cells (hASCs). By using a modified bioprinting process supplemented with a nozzle consisting of a single‐core channel and double‐sheath channels, we can achieve three different types of MTJ units, composed of muscle, tendon, and interface zones. Our results indicated that the bioprinted dECM‐based constructs induced hASCs to myogenic and tenogenic differentiation. In addition, a significantly higher MTJ‐associated gene expression was detected at the MTJ interface with a cell‐mixing zone than in the other interface models. Based on the results, the bioprinted MTJ model can be a potential platform for understanding the interaction between muscle and tendon cells, and even the bioprinting method can be extensively applied to obtain complex tissues.


| INTRODUCTION
In the human body, most tissues have unique physical and biological functions, with tissues with physically differing properties being present to perform specific biological or biomechanical functions. Furthermore, heterogeneous tissues are connected by specific interfaces for performing complicated functions. 1 The complex interfaces of orthopedic tissues can be divided into four types: cartilage-bone, ligament-bone, tendon-bone, and muscle-tendon interfaces. [2][3][4] Because most orthopedic tissue interfaces are biomechanical connections between relatively soft and hard materials, with the application of a high external strain to the region, injury, or damage caused by the stress concentration generally occurs in the transition region. 5 In particular, the myotendinous junction (MTJ) is important for transmitting force from a contracting muscle, through a tendon, to the bone, so that severe injuries, such as rotator cuff tears, Achilles tendon ruptures, and hamstring damage of the MTJ can occur as a result of repetitive and excessive loading or overtraining. 6 Specific MTJ structural interfaces, which are formed by the interaction between tenocytes and myotubes, consist of three typical regions: (1) elastic muscle, (2) stiff tendon, and (3) a complicated cell matrix. 7 The structure of the MTJ can be described as a tapered myofiber that can be entrenched in the tendon ECM.
Generally, the approach to heal an MTJ defect has involved the use of sutures, but this method has a high recurrence rate and induces the formation of fibrous scar tissue in the defective region. [8][9][10] To overcome this problem, MTJ regeneration using tissue engineering strategies has been pursued as an alternative and, in particular, research using functional biomaterials with structurally and biochemically biomimetic properties in the tissues of muscles and tendons and their interfaces have been the subject of several studies. [11][12][13][14] Although significant progress has been made in MTJ regeneration through the use of biomaterials and printing technologies, engineered biomaterials showing the unique physical/biological characteristics of MTJ are still required to obtain a perfect junction structure. Furthermore, most of the research has been focused on biomaterials and processing techniques to regenerate muscle and tendon tissues, although the MTJ function is a critical component in force transmission through the muscle-tendon, much less research has been conducted on the regeneration of the muscle and tendon interface. 15,16 In general, to regenerate MTJ substitutes, a typical scaffold-based strategy has been used. Using scaffold-based regeneration with synthetic or acellularized biomaterials and biomimetic hydrogels with or without cells, several advantageous factors, such as controllable pore geometry and physical properties, can be adapted as engineering substitutes. 17,18 However, the rigid scaffold can cause a stress-shielding effect on the cells for externally applied physical loading, eventually inducing low biological responses from the muscle and tendon cells. 5,7,19 In addition, an acellular structure that could be used to regenerate tendon tissue still has limitations including poor cell infiltration owing to the highly dense fibrous matrix. [20][21][22] In recent, electrospun nanofibers using polycaprolactone/chitosan/cellulose nanocrystal for regenerating tendon and ligament tissues were fabricated. 21 The human tendon-derived cells and human adipose-derived stem cells (hASCs), seeded on the nanofibers, have been organized into anisotropic tendon tissues, highly expressing tenogenesis-related genes. Although the successful induction to tenogenic differentiation was achieved, the cells infiltrated into the nanofibers have been observed after long culture period. Recently, to overcome scaffold limitations, a 3D bioprinting process has been used to obtain an MTJ construct that can mimic the physical structure and biofunctional characteristics of the native MTJ structure. 13 To serve mechanically functional constructs, highly elastic polyurethane and C2C12-containing hydrogel in the muscle tissue and stiff poly(ε-caprolactone) and NIH/3 T3-containing hydrogel in the tendon were applied. The hybrid construct demonstrated the characteristics of a mechanically heterogeneous MTJ scaffold, and the MTJ-related genes at the interface were expressed well.
Herein, we suggest an innovative biofabrication process to obtain an in vitro MTJ unit (muscle-MTJ-tendon). To obtain the construct, two typical biomaterials, namely, (1) decellularized extracellular matrixes (dECMs) derived from porcine muscle and tendon, and (2) type I collagen were used. To attain the MTJ unit, we adapted a 3D bioprinting process supplemented with a newly designed core-sheath nozzle in which a single-core channel and double-sheath channels were composed. The high weight fraction of collagen served as a mechanical supporter in the core, while in the sheath, two hASC-containing dECMbased bioinks were used as biological cell-differentiating niche components. Furthermore, to obtain aligned muscle and tendon tissue constructs, printing parameters, such as printing rates in the core and sheath and the collagen weight fraction in the core, were manipulated.
In addition, we fabricated three different types of MTJ constructs to evaluate which physical interfacing shape of muscle and tendon cells can best mimic the native MTJ structure by measuring MTJ-associated genes and mechanical properties.
The CM was changed every 2 days.
For the tendon bioink (T-bioink), tdECM was dissolved in 0.1 M acetic acid solution and mixed with 10Â DMEM at a ratio of 1:1.
For the collagen bioink, type I collagen sponge (MSbio, South Korea) was dissolved in 0.1 M acetic acid solution and mixed with 10Â DMEM at a ratio of 1:1. The neutralized collagen hydrogel (3 wt%) was mixed with hASCs (2 Â 10 7 cells/ml).
For the core channel, type I collagen sponge was dissolved in 0.1 M acetic acid solution and mixed with 10Â DMEM at a ratio of 1:1 to obtain collagen hydrogels of a range of concentrations (3, 4, 5, and 7 wt%).
To evaluate the myogenesis and tenogenesis of the stem cells, we performed two different cell cultures: (1) hASCs (5 Â 10 3 cells per cm 2 ) were seeded onto tissue culture plates coated with cell-free collagen, mdECM, and tdECM, and (2) three cell-laden bioinks (collagen, mdECM, and tdECM) with the cell-density (hASC, 2 Â 10 7 cells/ml) were cultured. The seeded cells and cell-laden bioinks were cultured using the CM under 5% CO 2 at 37 C. The CM was changed every 2 days.

| Bioprinting conditions
To fabricate the muscle, tendon, and MTJ structures, the prepared core collagen hydrogel, collagen bioink, M-bioink, and T-bioink were printed using a 3D printing system (DTR3-2210 T-SG; DASA Robot) supplemented with a modified core/sheath nozzle with one core channel and two sheath channels, and a pneumatic pressure dispenser (AD-3000C; Ugin-tech). The printing conditions, including the printing barrel temperature (20 C), working plate temperature (35-38 C), and printing rate (5 mm/s), were fixed. The printed structures were rinsed three times with DPBS and then cultured in CM under 5% CO 2 at 37 C. The CM was changed every 2 days.

| Characterization of bioinks and cellcontaining structures
The rheological properties (storage modulus, G) of the cell-containing bioinks were analyzed using a cone-and-plate geometry (diameter 40 mm, cone angle 4 , gap 150 μm) supplemented with a Bohlin Gemini HR Nano rotational rheometer (Malvern Instruments) conducted with a temperature sweep (strain 1%, frequency 1 Hz, temperature range 20-45 C, and ramping rate of 1 C/min). The gelation temperature of the bioinks was measured by observing the maximum point of G 0 (n = 3).
To measure the tensile properties of the printed structures (5 Â 15 Â 1 mm 3 ), a SurTA universal testing machine (Chemilab, South Korea) was used in the tensile testing mode (stretching rate: 0.05 mm/s). The Young's modulus was measured using the linear part (5%-10% strain) of the curves. All values are presented as a mean ± SD (n = 4). In addition, for the three types of MTJ units (2 Â 6.5 Â 1 mm 3 ), the load-displacement curves were plotted after culturing for 1, 28, and 42 days. The elastic stiffness was evaluated using the slope of the linear part ($1 mm displacement) of the loaddisplacement curves (n < 4).

| In vitro cellular responses
To analyze the cell viability after the fabrication process, the cells were

| Real-time polymerase chain reaction
To measure the gene expression levels, real-time polymerase chain reaction (qRT-PCR) was performed on cultured hASCs. The samples were treated with TRIzol reagent (Sigma-Aldrich) to isolate the total RNA, followed by measuring the purity and concentration of the isolated RNA using a spectrophotometer (FLX800T; Biotek). cDNA was synthesized using RNase-freeDNase-treated total RNA and Rev-erTraAceqPCR RT Master Mix (Toyobo Co., Ltd.). Finally, a StepOnePlus Real-Time PCR System (Applied Biosystems) and Thunderbird ® SYBER ® qPCR Mix (Toyobo Co., Ltd.) were used to perform qRT-PCR. The measured expression levels were normalized to the housekeeping gene glyceraldehyde 3-phosphate dehydrogenase. The gene expression levels of the hASCs seeded on the collagen-coated culture plates, hASC-containing collagen bioink, hASC-containing collagen structure, and type 1 MTJ models were set to onefold. Table 1 lists the gene-specific primers (Bionics) (n = 4).

| Statistical analyses
Statistical analyses were performed using SPSS software (SPSS, Inc.), conducted with the Student's t-test (comparing two groups) and single-factor ANOVA supplemented with Tukey's HSD post hoc test (comparing three or more groups). All quantitatively evaluated values are expressed as a mean ± SD, and values of p* < 0.05, p** < 0.01, and p*** < 0.001 were considered statistically significant. In this study, we used a mechanically enhanced cell-containing complex structure that can be applied to cell-containing hydrogels by using a modified core-sheath nozzle for the 3D bioprinting. To fabricate the structure, we designed a core-sheath nozzle in which singleand double-sheath channels were connected to the bioprinter  Figure 1c shows optical and immunofluorescence images (DAPI: blue, MHC: green, and TNMD: red) of the bioprinted MTJ unit at 28 days.

| Preparation of bioinks for MTJ unit
It has been well established that dECMs have unique biochemical/ physiological signals showing the native microenvironments, and thus they have been used as functional and bioactive materials. [35][36][37] To successfully create porcine-or bovine-derived dECMs, bioactive constituents such as collagen, elastin, GAGs, and intricate biomolecules should be present, but the cell components should be completely removed. 37 Here, we obtained dECMs derived from porcine skeletal muscle and Achilles tendon. As shown in the optical/immunofluorescence images  completely removed by the decellularization process. In particular, the DNA contents were below 1.1 and 5.8 ng/mg (muscle and tendon, respectively) in terms of dry weight, indicating that the decellularization was satisfactory because any value less than 50 ng/mg can be regarded as being a suitable value for the DNA content of dECM. 38 To show the bioactive feasibilities of the muscle and tendon dECMs, hASCs, specifically, mesenchymal stem cells derived from human adipose tissue were selected because they can be potentially differentiated into osteogenic, chondrogenic, myogenic, and even tenogenic lineages, by using biochemical, topographical, mechanical, and electrical stimulations. 39 In particular, because the differentiation of hASCs can be directly affected by biochemical components, the myogenic and tenogenic differentiation of hASCs was evaluated by staining with MHC for muscle and TNMD for tendon.
The induction of myogenesis and tenogenesis by dECMs were in good agreement with those of previous studies. 40,41 Those studies revealed that the hASCs containing muscle-derived dECM were fully differentiated into the myogenic lineage due to the biochemical components, transforming growth factor β1, insulin-like growth factor 1, and vascular endothelial growth factor, among others. 24,25,42,43 In addition, the tenogenic differentiation of hASCs could be clearly observed due to the increase in several integrin subunits and TGF-β/SMAD pathways in the hASCs, compared to the collagen control. 42

| Muscle and tendon structures bioprinted using dECMs and biophysical cues
The bioprinting process with a single core channel and double sheath channels was used to supply the pure collagen into the core region,  Based on the analysis of the printing parameters, we selected the flow rates in the core and sheath of 5.8 and 3.0 mm/s in the core and sheath, respectively.

| Fabrication of three MTJ constructs and their formation of an MTJ unit
To obtain the three different interface models (type 1: direct contact between muscle and tendon cells; type 2: artificially designed mixed zone of muscle and tendon cells, type 3: interdigitated contact between the muscle and tendon cells, as shown in Figure 5a  . 25 The biodegradation of the constructs measured over 21 days was presented in Figure S2A. In the results, the pure collagen was degraded at around 20.8%, but the M-and T-bioinks were more rapidly degraded by about 1.6-fold (M-bioink 34.9% and T-bioink 33.8%). In addition, we measured the biodegradability of the printed MTJ constructs without cells. As shown in Figure S2B, the degradation of the MTJ constructs (type 1 = 24.0%, type 2 = 24.8%, and type 3 = 24.3% at 21 days) was less than the constructs fabricated using writingreview and editing (lead).

DATA AVAILABILITY STATEMENT
The data that support the findings of this study are available from the corresponding author upon reasonable request.